Hydrogen evolution under photoheterotrophic and dark fermentative conditions by recombinant Rhodobacter sphaeroides containing the genes for fermentative pyruvate metabolism of Rhodospirillum rubrum

Rhodobacter sphaeroides KCTC 12085 is a natural isolate which shows a higher ability of photoheterotrophic H2 production compared with a laboratory strain R. sphaeroides 2.4.1. The H2 production by R. sphaeroides is observed only under light conditions. In order to render R. sphaeroides to produce H2 fermentatively as well, a 4.4-kb Rhodospirillum rubrum DNA encompassing a gene coding for pyruvate-formate lyase (PFL) with its putative maturation protein, and a 29.4-kb R. rubrum gene cluster including formate-hydrogen lyase (FHL) complex, its maturation proteins, a transcriptional activator for FHL, and Fe-only hydrogenase with its maturation proteins were cloned and mobilized into R. sphaeroides KCTC 12085. The recombinant R. sphaeroides evolved H2 during dark fermentative growth. The H2 production by the recombinant R. sphaeroides under photoheterotrophic conditions was elevated twofold to show 4 mol H2/mol of glucose compared with 2 mol H2/mol of glucose by its parental strain carrying empty vector. Interestingly, addition of hypophosphite, an inhibitor of PFL, to the recombinant R. sphaeroides under photoheterotrophic conditions still resulted in 3 mol H2/mol of glucose, suggestive of active H2 production not only by nitrogenase but also by Fe-only hydrogenase, which are insensitive to hypophosphite. The results further suggest the photoheterotrophic H2 production by FHL unless hypophosphite is present. Thus, the H2 production by the recombinant R. sphaeroides under photoheterotrophic conditions appears to be mediated by the concerted actions of FHL, Fe-only hydrogenase, and nitrogenase. The H2-evolving activity by the recombinant R. sphaeroides containing Fe-only hydrogenase of R. rubrum appears to depend on the presence its maturation proteins, suggestive of lack of such proteins in R. sphaeroides.